CN109477142B - Asymmetric templates and asymmetric methods of nucleic acid sequencing - Google Patents

Asymmetric templates and asymmetric methods of nucleic acid sequencing Download PDF

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CN109477142B
CN109477142B CN201780044275.7A CN201780044275A CN109477142B CN 109477142 B CN109477142 B CN 109477142B CN 201780044275 A CN201780044275 A CN 201780044275A CN 109477142 B CN109477142 B CN 109477142B
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nucleic acid
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CN109477142A (en
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R.陈
T.格托切
L.纳瓦罗
A.理查森
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F Hoffmann La Roche AG
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Abstract

The present invention is a novel method of making and using templates for nucleic acid sequencing. The templates include circular and linear templates with symmetric and asymmetric adaptors. The method includes utilizing the template in an asymmetric manner.

Description

Asymmetric templates and asymmetric methods of nucleic acid sequencing
Technical Field
The present invention relates to the field of nucleic acid sequencing, and more specifically to the preparation of circular templates for nucleic acid sequencing.
Background
The use of circular templates for sequencing is known in the art. For example, Pacific Biosciences use SMRTBell ™ adapters to generate such templates. See U.S. patent nos. 7,302,146 and 8,153,375. The circular single-stranded template has several advantages in sequencing while synthesizing: if the sequencing polymerase can perform rolling circle replication, the template will be read multiple times and the Watson and Crick strands will be read. Multiple reads of paired chains allow for more accurate consensus output. However, existing circular templates are designed such that two sequencing polymerases bind to each template. It is possible that two polymerases may interfere with each other and cause a stagnation or termination of synthesis, resulting in suboptimal sequencing data. The present invention improves upon the prior art to achieve more accurate sequencing reads.
Summary of The Invention
In some embodiments, the invention is a method of determining the sequence of a double-stranded target nucleic acid in a sample, comprising: contacting a sample comprising a double-stranded target nucleic acid with a hairpin adaptor molecule comprising a double-stranded stem region and a single-stranded loop region; ligating each end of the target nucleic acid molecule to a double stranded region of an adaptor molecule, thereby forming a circular adaptor molecule comprising the target nucleic acid and having a double stranded region and a proximal single stranded loop region and a distal single stranded loop region covalently linked to the double stranded region; contacting the sample with a defined concentration of a blocking oligonucleotide tethered to a solid support, wherein the blocking oligonucleotide is not extendable by a nucleic acid polymerase and is complementary to the proximal and distal single-stranded loop regions, thereby capturing the circular junction molecule on the solid support and blocking the proximal single-stranded loop region; contacting the sample with oligonucleotide primers complementary to proximal and distal single-stranded loop regions, thereby hybridizing the primers to the distal single-stranded loop region; extending the primer with a sequencing polymerase, thereby determining the sequence of the double stranded target nucleic acid. In some embodiments, the ends of the target nucleic acid and the adapter are blunt-ended. In some embodiments, the ends of the target nucleic acid and the adaptor are ligated by enzymatic treatment. The enzymatic treatment may be nucleotide addition and digestion of the target nucleic acid and the adaptor with a restriction endonuclease. The blocking oligonucleotide may be tethered to the solid support via a means selected from a covalent bond with a support molecule or a non-covalent bond with a support molecule. The non-covalent bond to the support molecule may be a specific interaction selected from the group consisting of: hybridization of biotin-streptavidin, antibody-antigen or blocking oligonucleotides to complementary oligonucleotides covalently or non-covalently attached to a solid support. The blocking oligonucleotide is not extendable by a nucleic acid polymerase. The blocking oligonucleotide may be rendered non-extendable by a nucleic acid polymerase by a chemical modification selected from the group consisting of 3' -H, 2' -phosphate and 3' -phosphate. The blocking oligonucleotide may have a modification that prevents binding of a nucleic acid polymerase. By virtue of being attached to the solid support via its 3' -end, the blocking oligonucleotide is not extendable by the nucleic acid polymerase. The blocking oligonucleotide may comprise one or more duplex stabilizing modifications, such as LNA, PNA and non-natural nucleotides. The blocker oligonucleotide comprises one or more modifications that block nuclease digestion, such as a phosphorothioate backbone. Each particle of the solid support is attached to a plurality of blocking oligonucleotides. In some embodiments, the method may further comprise the step of removing cyclic molecules not captured on the solid support. Sequencing may be single molecule sequencing, sequencing by synthesis, nanopore sequencing or tunnel recognition sequencing. In some embodiments, extending the primer is by a strand displacement polymerase or by rolling circle replication. In some embodiments, the double-stranded target nucleic acid is generated in vitro from a single-stranded target nucleic acid.
In some embodiments, the invention is a method of determining the sequence of a double-stranded target nucleic acid in a sample, comprising: contacting a sample comprising a double-stranded target nucleic acid with a mixture of first and second hairpin adaptor molecules, each of the first and second hairpin adaptor molecules comprising a double-stranded stem region and a first or second single-stranded loop region; ligating each end of the target nucleic acid molecule to a double stranded region of an adaptor molecule, thereby forming a circular adaptor molecule comprising the target nucleic acid and having a double stranded region and a first single stranded loop region at one end and a second single stranded loop region at the other end covalently linked to the double stranded region; contacting the sample with a defined concentration of a blocking oligonucleotide tethered to a solid support, wherein the blocking oligonucleotide is not extendable by a nucleic acid polymerase and is complementary to the first single-stranded loop region, thereby capturing the circular molecule on the solid support and blocking the first single-stranded loop region; contacting the sample with an oligonucleotide primer complementary to a second single stranded loop region, thereby hybridizing the primer to the second single stranded loop region; extending the primer with a sequencing polymerase, thereby determining the sequence of the double stranded target nucleic acid. The ends of the target nucleic acid and the adapter can be blunt-ended or made sticky by an enzymatic treatment that can be selected from the group consisting of nucleotide addition and digestion of the target nucleic acid and the adapter with a restriction endonuclease. In some embodiments, the blocking oligonucleotide is tethered to the solid support via a means selected from a covalent bond with a support molecule or a non-covalent bond with a support molecule. The non-covalent bond to the support molecule may be a specific interaction selected from the group consisting of: hybridization of biotin-streptavidin, antibody-antigen or blocking oligonucleotides to complementary oligonucleotides covalently or non-covalently attached to a solid support. The blocking oligonucleotide is not extendable by a nucleic acid polymerase due to, for example, a chemical modification selected from the group consisting of 3' -H, 2' -phosphate, and 3' -phosphate. The blocking oligonucleotide may have a modification that prevents binding of a nucleic acid polymerase. By virtue of being attached to the solid support via its 3' -end, the blocking oligonucleotide is not extendable by the nucleic acid polymerase. The blocking oligonucleotide may comprise one or more duplex stabilizing modifications, such as, for example, Locked Nucleic Acids (LNA), Peptide Nucleic Acids (PNA), and non-natural nucleotides. The blocker oligonucleotide may comprise one or more modifications that block nuclease digestion, such as a phosphorothioate backbone. In some embodiments, each particle of the solid support is attached to a plurality of blocking oligonucleotides. In some embodiments, the method further comprises removing nucleic acids not captured on the solid support. In some embodiments, the sequencing is single molecule sequencing, sequencing-by-synthesis, nanopore sequencing, or tunnel recognition sequencing. In some embodiments, extending the primer is by a strand displacement polymerase or by rolling circle replication. In some embodiments, the double-stranded target nucleic acid is generated in vitro from a single-stranded target nucleic acid.
In some embodiments, the invention is a method of determining the sequence of a double-stranded target nucleic acid in a sample, comprising: contacting a sample comprising a double-stranded target nucleic acid having two 5' -ends and two extendable 3' -ends with a terminal transferase, a single extendable nucleotide species, and providing means for controlling incorporation of nucleotides by the terminal transferase, extending the extendable 3' -ends by multiple units incorporating single nucleotides, thereby forming a linear-junction molecule comprising the target nucleic acid and having a double-stranded region and proximal and distal single-stranded homopolymeric regions; contacting the sample with a defined concentration of capture oligonucleotides tethered to a solid support, wherein the capture oligonucleotides are complementary to the proximal and distal single stranded homopolymer regions, thereby capturing the linear molecules on the solid support; contacting the sample with oligonucleotide primers complementary to proximal and distal single-stranded homopolymer regions, thereby hybridizing the primers to the distal single-stranded homopolymer regions; extending the primer with a sequencing polymerase, thereby determining the sequence of the double stranded target nucleic acid. The means for controlling nucleotide incorporation by terminal transferase is the presence of a terminator nucleotide species in the reaction at a ratio favorable for an extendible nucleotide, or the time of incorporation into the reaction. In some embodiments, the capture oligonucleotide is extendable, and the method further comprises extending the capture oligonucleotide to reach the end of the target nucleic acid. The method can further include the step of ligating the extended capture oligonucleotide to an end of the target nucleic acid to produce a continuous nucleic acid strand. The blocking oligonucleotide may be tethered to the solid support via a means selected from a covalent bond with a support molecule or a non-covalent bond with a support molecule. The non-covalent bond to the support molecule may be a specific interaction selected from the group consisting of: hybridization of biotin-streptavidin, antibody-antigen or blocking oligonucleotides to complementary oligonucleotides covalently or non-covalently attached to a solid support. In some embodiments, the blocking oligonucleotide comprises one or more duplex stabilizing modifications, such as Locked Nucleic Acids (LNA), Peptide Nucleic Acids (PNA), and non-natural nucleotides. In some embodiments, the blocker oligonucleotide comprises one or more modifications that block nuclease digestion, such as a phosphorothioate backbone. In some embodiments, each particle of the solid support is attached to a plurality of blocking oligonucleotides. In some embodiments, the method further comprises the step of removing nucleic acids not captured on the solid support. In some embodiments, each linear molecule has two different homopolymers produced by: blocking the proximal end of the target nucleic acid while extending the distal end with a mixture comprising a first non-terminator nucleotide, unbinding the proximal end and extending the proximal end with a mixture comprising a second non-terminator nucleotide. In some embodiments, the sequencing is single molecule sequencing, sequencing-by-synthesis, nanopore sequencing, or tunnel recognition sequencing. In some embodiments, the double-stranded target nucleic acid is generated in vitro from a single-stranded target nucleic acid.
In some embodiments, the invention is a composition for determining the sequence of a double-stranded target nucleic acid comprising: a circular junction molecule comprising a target nucleic acid attached at each end to an adaptor molecule comprising a double-stranded stem region and a single-stranded loop region, the circular junction molecule having a double-stranded region and proximal and distal single-stranded loop regions covalently attached to the double-stranded region; a blocking oligonucleotide tethered to a solid support, wherein the blocking oligonucleotide is not extendable by a nucleic acid polymerase and which hybridizes to a proximal single-stranded loop region, thereby capturing the circular adaptor molecule on the solid support and blocking the proximal single-stranded loop region; an oligonucleotide primer that hybridizes to the distal single-stranded loop region; and a sequencing polymerase.
In some embodiments, the invention is a composition for determining the sequence of a double-stranded target nucleic acid comprising: a linear junction molecule comprising a target nucleic acid and having a double-stranded region and proximal and distal single-stranded homopolymer regions; a capture oligonucleotide tethered to the solid support and hybridized to the proximal single stranded homopolymer region, thereby capturing the linear molecule on the solid support; an oligonucleotide primer that hybridizes to the distal single-stranded homopolymer region; and a sequencing polymerase.
Brief Description of Drawings
FIG. 1 shows a prior art method of preparing and sequencing a circular template. The capture probe hybridized to the adapter comprises the sequence shown as SEQ ID NO. 7 plus an additional sequence complementary to the hairpin region of the adapter.
Figure 2 shows an embodiment of the process of the invention. The capture probe hybridized to the adapter comprises the sequence shown as SEQ ID NO 8 plus an additional sequence complementary to the hairpin region of the adapter.
FIG. 3 shows details of the nucleotide sequence details of the structure illustrated in FIG. 2. Hairpin adaptor A comprises the sequence shown in SEQ ID NO 1. Hairpin adaptor B comprises the sequence shown in SEQ ID NO 2. The capture probe is shown in SEQ ID NO. 4. The sequencing primer is shown as SEQ ID NO. 5.
FIG. 4 shows a symmetric linear template with homopolymer (SEQ ID NO: 9).
Figure 5 shows a symmetric linear template with asymmetric loading of a sequencing polymerase. (homopolymer shown in SEQ ID NO: 9.)
FIG. 6 shows details of nucleotide sequence details of another embodiment of the structure illustrated in FIG. 2. Hairpin adaptor A comprises the sequence shown in SEQ ID NO 1. Hairpin adaptor C comprises the sequence shown in SEQ ID NO 3. The bead-attached capture sequence is shown in SEQ ID NO 10. The sequencing primer is shown as SEQ ID NO. 5.
FIG. 7 shows details of the nucleotide sequence details of another embodiment of the structure illustrated in FIG. 2. Hairpin adaptor A comprises the sequence shown in SEQ ID NO 1. Hairpin adaptor B comprises the sequence shown in SEQ ID NO 2. The capture probe/sequencing primer is shown in SEQ ID NO 6.
FIGS. 8A-8F show a serial assembly workflow for preparing sequencing libraries for sequencing according to the methods of the invention. The workflow was started with oligo-DT capture beads (fig. 8A). The capture probe (SEQ ID NO:4) was added to the capture beads to form capture probe-capture bead complexes (FIG. 8B). A circular sequencing template comprising a double-stranded target nucleic acid molecule and two hairpin adaptors (hairpin adaptors A and B comprising SEQ ID NOS: 1 and 2, respectively) was contacted with the capture probe-capture bead complex (FIG. 8C) to form a complex of capture beads, capture probes, and circular sequencing template. Then, a sequencing primer (SEQ ID NO:5) (FIG. 8D) and a sequencing polymerase (FIG. 8E) were added. Figure 8F shows analysis of the components of the assembled sequencing library when components are added as shown in figures 8A-8E. All three were detected in the final assembled complex only when all included beads, capture probes and library molecules.
Detailed Description
Definition of
The term "sample" refers to any composition that contains or is assumed to contain a target nucleic acid. This includes samples of tissues or fluids isolated from an individual, such as skin, plasma, serum, spinal fluid, lymph fluid, synovial fluid, urine, tears, blood cells, organs, and tumors, as well as samples of in vitro cultures established from cells taken from an individual, including formalin-fixed paraffin-embedded tissue (FFPET) and nucleic acids isolated therefrom. The sample may also include cell-free material, such as a cell-free blood fraction containing cell-free dna (cfdna) or circulating tumor dna (ctdna).
The term "nucleic acid" refers to a polymer of nucleotides (e.g., natural and non-natural ribonucleotides and deoxyribonucleotides), including DNA, RNA, and subsets thereof, such as cDNA, mRNA, and the like. Nucleic acids can be single-stranded or double-stranded and will typically contain 5'-3' phosphodiester linkages, although in some cases, nucleotide analogs can have other linkages. Nucleic acids may include naturally occurring bases (adenosine, guanosine, cytosine, uracil and thymidine) as well as non-natural bases. Some examples of non-natural bases are included in, for example, Seela et al, (1999)Helv. Chim. Acta1640, respectively, to the corresponding compounds described in 82: 1640. The non-natural base may have a specific function, for example, increasing the stability of the nucleic acid duplex, inhibiting nuclease digestion or blocking primer extension or strand polymerization.
The terms "polynucleotide" and "oligonucleotide" are used interchangeably. Polynucleotides are single-stranded or double-stranded nucleic acids. Oligonucleotides are a term sometimes used to describe shorter polynucleotides. The oligonucleotide may be composed of at least 6 nucleotides or about 15-30 nucleotides. Oligonucleotides are prepared by any suitable method known in the art, for example, by methods involving direct chemical synthesis, as described below: narang et al (1979)Meth. Enzymol.68:90-99, Brown et al (1979)Meth. Enzymol.68:109-Tetrahedron Lett.22:1859-J. Am. Chem. Soc. 103:3185-3191。
The term "primer" refers to a single-stranded oligonucleotide that hybridizes to a sequence in a target nucleic acid (the "primer binding site") and is capable of acting as a point of initiation of synthesis along a complementary strand of nucleic acid under conditions suitable for such synthesis.
The term "adaptor" means a nucleotide sequence that can be added to another sequence in order to input additional properties into the sequence. Adapters are typically oligonucleotides that may be single-stranded or double-stranded, or may have both single-stranded and double-stranded portions.
The term "ligation" refers to a condensation reaction that joins two nucleic acid strands in which the 5 '-phosphate group of one molecule reacts with the 3' -hydroxyl group of the other molecule. Ligation is typically an enzymatic reaction catalyzed by a ligase or a topoisomerase. Ligation may join two single strands to produce a single-stranded molecule. Ligation may also join two strands (each of which belongs to a double-stranded molecule), thus joining two double-stranded molecules. Ligation may also join two strands of a double-stranded molecule to two strands of another double-stranded molecule, thus joining the two double-stranded molecules. Ligation can also join the two ends of a strand within a double-stranded molecule, thus repairing the nicks in the double-stranded molecule.
The term "barcode" refers to a nucleic acid sequence that can be detected and identified. Barcodes can be incorporated into other nucleic acids. Barcodes are sufficiently long, e.g., 2, 5, 10 nucleotides, such that in a sample, the nucleic acids incorporated into the barcode can be distinguished or grouped according to the barcode.
The term "multiplex identifier" or "MID" refers to a barcode that identifies the source of a target nucleic acid (e.g., a sample from which the nucleic acid is derived). All or substantially all target nucleic acids from the same sample will share the same MID. Target nucleic acids from different sources or samples can be mixed and sequenced simultaneously. Using MID, sequence reads can be assigned to individual samples from which target nucleic acids are derived.
The term "unique molecular identifier" or "UID" refers to a barcode that identifies a nucleic acid to which it is attached. All or substantially all target nucleic acids from the same sample will have different UIDs. All or substantially all progeny (e.g., amplicons) derived from the same original target nucleic acid will share the same UID.
The terms "universal primer" and "universal priming binding site" or "universal priming site" refer to primers and primer binding sites that are present (typically, added in vitro) in different target nucleic acids. The universal priming site is added to the plurality of target nucleic acids using adapters or using target-specific (non-universal) primers with universal priming sites in the 5' -portion. The universal primer can bind to the universal priming site and direct primer extension from the universal priming site.
As used herein, the term "target sequence", "target nucleic acid" or "target" refers to a portion of a nucleic acid sequence in a sample to be detected or analyzed. The term target includes all variants of the target sequence, e.g., one or more mutant variants and wild-type variants.
The term "sequencing" refers to any method of determining the sequence of nucleotides in a target nucleic acid.
The present invention provides a method for determining the sequence of a double-stranded target nucleic acid in a sample, comprising:
(a) contacting a sample comprising a double-stranded target nucleic acid with a hairpin adaptor molecule comprising a double-stranded stem region and a single-stranded loop region;
(b) ligating each end of the target nucleic acid molecule to a double stranded region of an adaptor molecule, thereby forming a circular adaptor molecule comprising the target nucleic acid and having a double stranded region and a proximal single stranded loop region and a distal single stranded loop region covalently linked to the double stranded region;
(c) contacting the sample with a defined concentration of a blocking oligonucleotide tethered to a solid support, wherein the blocking oligonucleotide is not extendable by a nucleic acid polymerase and is complementary to the proximal single-stranded loop region, thereby capturing the circular junction molecule on the solid support and blocking the proximal single-stranded loop region;
(d) contacting the sample with an oligonucleotide primer complementary to a distal single-stranded loop region, thereby hybridizing the primer to the distal single-stranded loop region;
(e) extending the primer with a sequencing polymerase, thereby determining the sequence of the double stranded target nucleic acid.
Alternatively, the present invention provides a method for determining the sequence of a double-stranded target nucleic acid in a sample, comprising:
(a) contacting a sample comprising a double-stranded target nucleic acid with a mixture of first and second hairpin adaptor molecules, each of the first and second hairpin adaptor molecules comprising a double-stranded stem region and a first or second single-stranded loop region;
(b) ligating each end of the target nucleic acid molecule to a double stranded region of an adaptor molecule, thereby forming a circular adaptor molecule comprising the target nucleic acid and having a double stranded region and a first single stranded loop region at one end and a second single stranded loop region at the other end covalently linked to the double stranded region;
(c) contacting the sample with a defined concentration of a blocking oligonucleotide tethered to a solid support, wherein the blocking oligonucleotide is not extendable by a nucleic acid polymerase and is complementary to the first single-stranded loop region, thereby capturing the circular molecule on the solid support and blocking the first single-stranded loop region;
(d) contacting the sample with an oligonucleotide primer complementary to a second single stranded loop region, thereby hybridizing the primer to the second single stranded loop region;
(e) extending the primer with a sequencing polymerase, thereby determining the sequence of the double stranded target nucleic acid.
After step (c), the bound molecules not captured on the solid support may be removed.
The ends of the target nucleic acid and the adapter can be flattened or sticky by enzymatic treatment. The enzymatic treatment may be selected from the group consisting of nucleotide addition and digestion of the target nucleic acid and the adaptor with a restriction endonuclease. The blocking oligonucleotide may be tethered to the solid support via a means selected from a covalent bond with a support molecule or a non-covalent bond with a support molecule. The non-covalent bond to the support molecule may be a specific interaction selected from the group consisting of: hybridization of biotin-streptavidin, antibody-antigen or blocking oligonucleotides to complementary oligonucleotides covalently or non-covalently attached to a solid support. The blocking oligonucleotide may be non-extendable by a nucleic acid polymerase and may be rendered non-extendable by a chemical modification selected from the group consisting of 3' -H, 2' -phosphate, and 3' -phosphate. The blocking oligonucleotide may have a modification that prevents binding of a nucleic acid polymerase. By virtue of being attached to the solid support via its 3' -end, the blocking oligonucleotide is not extendable by the nucleic acid polymerase. The blocking oligonucleotide may comprise one or more duplex stabilizing modifications, which may be selected from the group consisting of Locked Nucleic Acids (LNA), Peptide Nucleic Acids (PNA) and non-natural nucleotides. The blocker oligonucleotide may comprise one or more modifications that block nuclease digestion, such as a phosphorothioate backbone. The solid support may be comprised of particles, and each particle may be attached to a plurality of blocking oligonucleotides.
Sequencing may be single molecule sequencing, sequencing by synthesis, nanopore sequencing or tunnel recognition sequencing. Primer extension can be performed by strand displacement polymerase and/or by rolling circle replication. The double stranded target nucleic acid can be generated in vitro from a single stranded target nucleic acid.
The present invention also provides a method for determining the sequence of a double-stranded target nucleic acid in a sample, comprising:
(a) contacting a sample comprising a double-stranded target nucleic acid having two 5 '-ends and two extendable 3' -ends with a terminal transferase and a single extendable nucleotide species, and providing means for controlling incorporation of nucleotides by the terminal transferase;
(b) extending the extendable 3' -end by a plurality of units incorporating a single nucleotide, thereby forming a linear junction molecule comprising a target nucleic acid and having a double-stranded region, a proximal single-stranded homomeric region and a distal single-stranded homomeric region;
(c) contacting the sample with a defined concentration of capture oligonucleotides tethered to a solid support, wherein the capture oligonucleotides are complementary to the proximal and distal single stranded homopolymer regions, thereby capturing the linear molecules on the solid support;
(d) contacting the sample with oligonucleotide primers complementary to proximal and distal single-stranded homopolymer regions, thereby hybridizing the primers to the distal single-stranded homopolymer regions;
(e) extending the primer with a sequencing polymerase, thereby determining the sequence of the double stranded target nucleic acid.
After step (c), nucleic acids not captured on the solid support may be removed.
The means for controlling the incorporation of nucleotides by the terminal transferase can be the presence of a terminator nucleotide species in the reaction at a ratio favorable for an extendible nucleotide, or the time of incorporation into the reaction.
If the capture oligonucleotide is not extendable, the method may further comprise, after step (c), extending the capture oligonucleotide to reach the end of the target nucleic acid. The method can then further include the step of ligating the extended capture oligonucleotide to an end of the target nucleic acid to produce a continuous nucleic acid strand.
The blocking oligonucleotide may be tethered to the solid support via a means selected from a covalent bond with a support molecule or a non-covalent bond with a support molecule. The non-covalent bond to the support molecule may be a specific interaction selected from the group consisting of: hybridization of biotin-streptavidin, antibody-antigen or blocking oligonucleotides to complementary oligonucleotides covalently or non-covalently attached to a solid support. The blocking oligonucleotide may comprise one or more duplex stabilizing modifications, such as Locked Nucleic Acids (LNA), Peptide Nucleic Acids (PNA) or non-natural nucleotides. The blocker oligonucleotide may comprise one or more modifications that block nuclease digestion, such as a phosphorothioate backbone. The solid support may be comprised of particles, and each particle may be attached to a plurality of blocking oligonucleotides.
Each linear molecule may have two different homopolymers produced by: blocking the proximal end of the target nucleic acid while extending the distal end with a mixture comprising a first non-terminator nucleotide, unbinding the proximal end and extending the proximal end with a mixture comprising a second non-terminator nucleotide.
Sequencing may be single molecule sequencing, sequencing by synthesis, nanopore sequencing or tunnel recognition sequencing. The double stranded target nucleic acid can be generated in vitro from a single stranded target nucleic acid. In some embodiments, the invention is a method of converting a double-stranded target nucleic acid into a circular locked strand template structure that can be used in sequencing. The use of circular templates is known in the art and has several advantages in sequencing-by-synthesis applications. See U.S. patent nos. 7,302,146 and 8,153,375 and fig. 1. If a strand displacing polymerase is used, it will participate in rolling circle replication, i.e., successive displacement of the nascent strand, and multiple rounds of replication of the circular template. The ability to sequence the target multiple times (read through) and compare the Watson and Crick strands linked to the target nucleic acid in a circular structure allows for the generation of error-free or low-error consensus sequences.
However, existing circular templates are designed with adapters attached to both ends of the target nucleic acid. (FIG. 1). Each adapter has a binding site for a sequencing primer that allows two sequencing primers and two sequencing DNA polymerases to bind to each circular template. Once the sequencing reaction has been initiated, the two polymerases may interfere with each other and cause a lag or termination of synthesis, reducing the read length and yield of sequencing data. This is particularly problematic for shorter templates. In some applications, such as single-molecule side-by-synthesis sequencing, the presence of two polymerases (e.g., nanopores or zero-molecular waveguides (ZMWs)) per detection spot will degrade the quality of the sequencing data.
The present invention improves upon the prior art by ensuring that only a single sequencing polymerase binds to each sequencing template and synthesis proceeds in only one direction. (FIG. 2). The present invention is a novel method that can increase sequencing quality, read length, and efficiency. In embodiments of the invention, a double stranded target nucleic acid is conjugated to two adaptors, one at each end of the molecule. Each adapter sequence has a primer binding site (e.g., a universal primer binding site) at which sequencing is initiated. The resulting adaptor molecule is captured at one end while the second end remains available for sequencing polymerase. (FIG. 2).
The invention is a method of generating a template for sequencing a target nucleic acid or a target nucleic acid library. In some embodiments, in a first step, a target nucleic acid is contacted with a stem-loop adaptor molecule comprising a double stranded stem region and a single-stranded loop region. Ligating each end of the target nucleic acid to an adaptor, thereby forming a junction molecule. If the adaptor has a stem-loop structure, the resulting adaptor molecule has a double-stranded region (comprising the target nucleic acid) and proximal and distal single-stranded loop regions covalently linked to the double-stranded region. The adaptor molecule is a topologically closed circular molecule consisting of a single continuous chain. In some embodiments, the unligated adapters and unligated target nucleic acids are removed from the sample prior to further processing.
In some embodiments, all adaptor molecules are the same. In other embodiments, there is a mixture of two adaptor molecules. In the case of an equal mixture of two adaptors (e.g., a and B), 50% of the adaptor molecules will have the desired asymmetric adaptor structure (e.g., a-B).
The adaptor molecule is then contacted with a defined concentration of blocking oligonucleotide tethered to a solid support. Blocking oligonucleotides may also be referred to as "capture oligonucleotides" because they may be used to capture the conjugated molecule, e.g., in solution or on a solid support. Capture may comprise simply forming a hybridization complex with the capture oligonucleotide, or capture may comprise capture on a solid support to which the oligonucleotide may be tethered. The blocking oligonucleotide at a defined concentration serves to ensure that only one of the two adaptor-ends is captured or blocked, while the other adaptor-end can still enter into further enzymatic steps. In embodiments with two different adaptors per adapter molecule, the capture oligonucleotide is complementary to only one adaptor, and not the other, ensuring that only one adaptor-end is captured. In embodiments where each adaptor molecule has the same adaptor at both ends, the blocking oligonucleotide is complementary to the adaptor. The use of a defined concentration of blocking oligonucleotide ensures that only one adapter-end is captured, while the other adapter-end is still accessible. For convenience, in the present disclosure, the captured adaptor-end is designated as proximal, while the free adaptor-end is designated as distal.
As is evident from the next step described below, the blocking capture oligonucleotide must be non-extendable at its 3' -end by a nucleic acid polymerase. The capture oligonucleotide is at least partially complementary to the proximal adaptor-end and hybridizes to the single stranded portion of the adaptor-end, thereby blocking the adaptor-end. Because the 3' -end of the blocking oligonucleotide is not extendible, it cannot serve as the start of chain polymerization in the presence of a nucleic acid polymerase. Optionally, uncaptured conjugated molecules may be removed before the next step.
Next, the sample is contacted with an oligonucleotide primer complementary to the primer binding site in the single-stranded region of the adaptor. Because the proximal end is blocked, only the distal end is available for primer binding and hybridization. The primer can be extended with a sequencing polymerase, thereby determining the sequence of the double-stranded target nucleic acid. The sequencing is sequencing-by-synthesis, including single molecule sequencing or any sequencing of nucleic acids or nucleic acid derivatives. The sequencing techniques may comprise a PacBio RS system, a nanopore sequencing system, or a tunnel recognition sequencing system, or any sequencing system in which it is possible and desirable to read the template continuously.
In some embodiments, primer extension is performed by a strand displacement polymerase. In some embodiments, primer extension occurs via rolling circle replication, such that each template can be read multiple times by a single polymerase.
In some embodiments of the method, the adapter flanking the end of the target nucleic acid is a single stranded homopolymer region. Rather than ligating adapters to the ends of the target nucleic acid as described in the embodiments above, the extendable 3' -end of the target nucleic acid is extended by contacting the sample with a suitable enzyme and a single extendable nucleotide species. Suitable enzymes are template-independent DNA polymerases such as, for example, terminal deoxynucleotidyl transferase (TdT). The sequential addition of one nucleotide species generates a homopolymer region on both sides of the target nucleic acid. In some embodiments, the methods comprise means for controlling the size of the homopolymer region. Control can be achieved by the reaction in the presence of the ratio of favorable extendible nucleotide terminator nucleotide species or incorporation reaction time to complete. Suitable enzymes may be terminal transferases.
In this embodiment, the adaptor molecule is a linear molecule comprising the target nucleic acid and having a double stranded region and proximal and distal single stranded homopolymer regions. In this embodiment, the capture oligonucleotide may be complementary to the homopolymer region and capable of capturing the single stranded homopolymer region, thereby capturing the linear adaptor molecule on the solid support. The region of the homopolymer trapped on the solid support is referred to as the proximal region, and the free region is the distal region. As in other embodiments of the invention, the sequencing primer is capable of binding to and hybridizing to a free distal homopolymer region and priming primer extension. In some embodiments, the method further comprises the step of removing nucleic acids not captured on the solid support.
In some embodiments, each linear molecule has two different homopolymers that are generated by sequentially blocking one end of a target nucleic acid while extending the other end with different nucleotides. In such embodiments, the capture oligonucleotide and the primer are complementary to the homopolymer on opposite sides of the adaptor molecule.
The present invention includes detecting a target nucleic acid in a sample. In some embodiments, the sample is derived from a subject or patient. In some embodiments, the sample may comprise a fragment of a solid tissue or solid tumor derived from the subject or patient, e.g., by biopsy. The sample can also include a bodily fluid (e.g., urine, sputum, serum, plasma or lymph fluid, saliva, sputum, sweat, tears, cerebrospinal fluid, amniotic fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, cyst fluid, bile, gastric fluid, intestinal fluid, and/or stool sample). The sample may comprise whole blood or a blood fraction in which tumor cells may be present. In some embodiments, the sample, particularly a liquid sample, may comprise cell-free material, such as cell-free DNA or RNA, including cell-free tumor DNA or tumor RNA. In some embodiments, the sample is a cell-free sample, such as a cell-free blood-derived sample, in which cell-free tumor DNA or tumor RNA is present. In other embodiments, the sample is a cultured sample, e.g., a culture or culture supernatant, containing or suspected of containing an infectious agent or a nucleic acid derived from an infectious agent. In some embodiments, the infectious agent is a bacterium, protozoan, virus, or mycoplasma.
A target nucleic acid is a target nucleic acid that may be present in a sample. In some embodiments, the target nucleic acid is a gene or gene fragment. In other embodiments, the target nucleic acid contains genetic variants, e.g., polymorphisms, including single nucleotide polymorphisms or single nucleotide variants (SNPs or SNVs), or genetic rearrangements that result in, e.g., gene fusions. In some embodiments, the target nucleic acid comprises a biomarker. In other embodiments, the target nucleic acid is characteristic of a particular organism, e.g., a characteristic that aids in identifying a pathogenic organism or pathogenic organism, e.g., drug sensitivity or drug resistance. In still other embodiments, the target nucleic acid is characteristic of a human subject, e.g., an HLA or KIR sequence that defines the subject's unique HLA or KIR genotype. In still other embodiments, all sequences in the sample are target nucleic acids, e.g., in shotgun genome sequencing.
In one embodiment of the invention, a double stranded target nucleic acid is converted into a template configuration of the invention. In some embodiments, the target nucleic acid is present in nature in a single-stranded form (e.g., RNA, including mRNA, microRNA, viral RNA, or single-stranded viral DNA). Converting the single-stranded target nucleic acid to a double-stranded form to accomplish the additional step of the claimed method. Although longer target nucleic acids may be required to achieve longer reads in some applications, longer target nucleic acids may be fragmented. In some embodiments, the target nucleic acid is naturally fragmented, e.g., circulating cell-free DNA (cfdna) or chemically degraded DNA, such as that found in a stored sample.
In some embodiments of the invention, the adaptor molecule is ligated to the target nucleic acid. The ligation may be blunt-ended or more efficient cohesive-ended. The target nucleic acid or adapter ends can be flattened by strand filling, i.e., by extending the 3 '-ends with a DNA polymerase to eliminate the 5' -overhang. In some embodiments, blunt-ended adaptors and target nucleic acids can be made cohesive by adding a single nucleotide to the 3 '-end of the adaptor and a single complementary nucleotide to the 3' -end of the target nucleic acid, for example, by a DNA polymerase or terminal transferase. In still other embodiments, the adapter and the target nucleic acid can be made cohesive ends (overhangs) by digestion with a restriction endonuclease. The latter option is more advantageous for known target sequences known to contain restriction enzyme recognition sites. In each of the above embodiments, the adaptor molecule may be designed to obtain the desired end (blunt end, single base extension, or multi-base overhang) by synthetic adaptor oligonucleotides described further below. In some embodiments, additional enzymatic steps may be required to complete the ligation. In some embodiments, polynucleotide kinases can be used to add 5' -phosphate to a target nucleic acid molecule and an adaptor molecule.
The invention includes the use of adaptor molecules to be ligated to one or both ends of the target nucleic acid. In some embodiments, the adaptor is single-stranded of a nucleic acid employing a stem-loop secondary structure comprising at least one double-stranded region and at least one single-stranded region. The double stranded region comprises a region having at least partial self complementarity ensuring stability of the secondary structure under the reaction conditions used herein. In some embodiments, the adaptor molecule is an artificial sequence synthesized in vitro. In other embodiments, the adaptor molecule is a naturally occurring sequence that is known to have the desired secondary structure, synthesized in vitro. In still other embodiments, the adaptor molecule is an isolated naturally occurring molecule or an isolated non-naturally occurring molecule.
In some embodiments, the adaptor comprises at least one double stranded region and at least one single stranded region. In some embodiments, the adaptor forms a stem-loop secondary structure having at least one double-stranded stem and at least one single-stranded loop. In some embodiments, a double-stranded stem is used to ligate to a double-stranded target nucleic acid. In other embodiments, the single stranded portion of the adapter is ligated to the single stranded portion of the target nucleic acid. In some embodiments, the ligation of single-stranded nucleic acids is performed using a splint oligonucleotide, see, e.g., U.S. application publication No. 20120003657. In other embodiments, the ligation of single-stranded nucleic acids or portions of single-stranded nucleic acids is performed using 5 '-and 3' -terminal single-stranded regions (overhangs), see, e.g., U.S. application publication No. 20140193860.
In some embodiments, the adapter comprises one or more barcodes: multiplex sample id (MID), Unique Id (UID), or a combination of UID and MID. In some implementations, a single barcode is used as both the UID and the MID.
In some embodiments, the adaptors comprise primer binding sites for universal primers (e.g., universal sequencing primers). In some embodiments, the adaptor comprises a binding site for a capture oligonucleotide. In some embodiments, the adapter used in the methods of the invention is a mixture of an adapter comprising a binding site for a primer and an adapter comprising a binding site for a capture oligonucleotide.
In some embodiments, the invention includes the use of capture oligonucleotides. In some embodiments, the capture oligonucleotide is directly bound to the solid support. In this embodiment, the capture oligonucleotide comprises a binding moiety at one end and a free end at the other end. The capture oligonucleotide is tethered to a solid support (e.g., bead, microsphere) via a binding moiety. In some embodiments, the tethered end of the oligonucleotide comprises biotin, and the solid support is coated with streptavidin. In other embodiments, the tethered end of the oligonucleotide comprises a capture molecule, and the solid support comprises an antibody specific for the capture molecule. For example, digoxigenin and anti-digoxigenin antibodies can be used.
In some embodiments, the capture oligonucleotide is not directly bound to the solid support, but is hybridized to another oligonucleotide ("bead oligonucleotide") that is directly attached to the solid support by any of the methods described above. The capture oligonucleotide and the bead oligonucleotide share at least one region of complementarity. For example, the bead oligonucleotide may comprise a homopolymer of dT (oligo-dT) and a portion of the capture oligonucleotide is a homopolymer of dA (oligo-dA). (FIGS. 3, 6 and 7).
In some embodiments, the blocking oligonucleotide is not extendable at the 3' -terminus by a nucleic acid polymerase. The 3' -end can be rendered non-extendable by chemical modification. For example, 3' -H, 2' -phosphate and 3' -phosphate are such modifications. The blocking oligonucleotide may have a modification that prevents binding of the nucleic acid polymerase, such as a bulky adduct that sterically blocks the 3' -terminus. The blocking oligonucleotide may be rendered non-extendable by virtue of being attached to the solid support via its 3' -end. The blocker oligonucleotide may also comprise one or more modifications that block nuclease digestion, such as a phosphorothioate backbone.
In some embodiments, the oligonucleotide comprises a free 5 '-end and has a 3' -end tethered to a solid support. In other embodiments, the oligonucleotide comprises a free 3 '-end and has a 5' -end tethered to a solid support. At least a portion of the free 5 '-end or 3' -end is complementary to a sequence in the adaptor. In some embodiments, the free end is complementary to a single stranded portion of the adaptor, e.g., complementary to a loop structure. Via the complementary portion, a capture oligonucleotide tethered to the solid support hybridizes to a junction molecule comprising the target nucleic acid attached to at least one adapter. The capture oligonucleotide may comprise one or more modifications that stabilize the hybrid. In some embodiments, the modifications are selected from the group consisting of Locked Nucleic Acids (LNAs), Peptide Nucleic Acids (PNAs), non-natural nucleotides such as 7-azapurines (e.g., 7-azaguanine, 7-azaadenine, etc.), pyrazolo [3,4-d ] pyrimidines, propynyl-dN (e.g., propynyl-dU, propynyl-dC, etc.), and the like, as described, for example, in U.S. patent No. 5,990,303.
In some embodiments, the method involves generating a conjugated molecule. The adapter molecule comprises a double stranded target nucleic acid linked to one or more adapter molecules. In some embodiments, the adaptor molecule is a topologically circular (closed) single-stranded comprising a double-stranded region (comprising the target nucleic acid) flanked at each end by a closed-stranded region (comprising the adaptor sequence).
In some embodiments, the adapter molecules have identical ends, i.e., are ligated to two identical adaptors. In other embodiments, the adaptor molecule comprises different ends, each end being linked to a different adaptor molecule. In some embodiments, the adaptor molecule is a mixture of two types of adaptors (e.g., a and B). The sample then contains a mixture of adapter molecules with adapters AA, AB and BB in a specific ratio. In some embodiments, an equal ratio of a and B is used. 50% of the resulting conjugated molecules will have the desired structure A-B. 25% will be A-A, i.e.there will be no binding site for the sequencing primer, and 25% will be B-B, as used for the two sites of the sequencing primer in the prior art. In such cases, the present invention would provide an improvement over the prior art in that the conjugated molecules (a-B) of 2/3 would be processed according to an improved method to generate an improved read.
In some embodiments, the adaptor molecule is a linear molecule comprising a double stranded region comprising a target nucleic acid flanked at each end by an adaptor or similar sequence. In some embodiments, the adaptor is a linear double stranded molecule. In other embodiments, the adaptor may be a linear single stranded molecule. In still other embodiments, the double-stranded region comprising the target nucleic acid is flanked by one or two homopolymers.
In some embodiments, the invention utilizes enzymes. The enzymes include DNA polymerases (including sequencing polymerases), DNA ligases, and terminal transferases.
In some embodiments, the DNA polymerase has strand displacement activity and does not have 5' -3-exonuclease activity. In some embodiments, Phi29 polymerase and its derivatives are used. See U.S. patent nos. 5,001,050, 5576204, 7858747 and 8921086.
In some embodiments, the invention also utilizes DNA ligase. In some embodiments, T4 DNA ligase or e.coli DNA ligase is used.
In some embodiments, the invention also utilizes a template-independent DNA polymerase, such as a terminal transferase. In some embodiments, the invention uses a mammalian terminal transferase.
In some embodiments, the invention is a composition for determining the sequence of a double-stranded target nucleic acid comprising: a circular adaptor molecule comprising a target nucleic acid and having a double stranded region and a proximal and a distal single stranded loop region covalently linked to the double stranded region, the proximal region being hybridized to a blocking oligonucleotide that is not extendable by a nucleic acid polymerase. The blocking oligonucleotide may be tethered to a solid support. In some embodiments, the composition further comprises oligonucleotide primers complementary to the proximal and distal single-stranded loop regions and optionally a nucleic acid polymerase.
In some embodiments, the invention is a composition for determining the sequence of a double-stranded target nucleic acid comprising: a linear junction molecule comprising a target nucleic acid and having a double-stranded region and proximal and distal single-stranded homopolymer regions, wherein the proximal homopolymer region is hybridized to a blocking oligonucleotide that is not extendable by a nucleic acid polymerase. The blocking oligonucleotide may be tethered to a solid support. In some embodiments, the composition further comprises oligonucleotide primers complementary to the proximal and distal single-stranded loop regions and optionally a nucleic acid polymerase.
Examples
Example 1 (predictive) preparation of symmetrically adjusted circular adaptor molecules for asymmetric sequencing polymerases Sequencing of the load
In this experiment, a double-stranded target DNA was obtained. The DNA is fragmented to the appropriate size in vitro or naturally fragmented. Adaptors are hairpin molecules having a double stranded portion and a loop portion. The same adapters were ligated to each end of the target DNA to generate adaptor molecules as described in Pacific Biosciences Template conjugation and Sequencing Guide (2012) Pacific Biosciences of California, Inc. and U.S. Pat. No. 8,153,375. See fig. 2A. The capture oligonucleotide is complementary to the single stranded portion of the adaptor molecule (FIG. 2B). The capture oligonucleotides also contained a poly-dA moiety complementary to a poly-dT oligonucleotide bound to polystyrene coated magnetic beads (DynaBeads, ThermoFisher, Waltham, Mass.) (see FIG. 2A). The capture oligonucleotide has several LNA bases that stabilize the complex with the adapter (see fig. 2A and 2B). Thus, another adaptor can be used for polymerase loading and sequencing initiation. The sequencing primer is complementary to the single-stranded portion of the adapter, and the sequencing polymerase is capable of extending the primer, thereby performing a sequencing reaction. The components were added in the order shown in figure 2.
An excess of the adaptor molecules compared to the concentration of bead-bound capture oligonucleotides (high library: bead ratio) ensures that a sufficient amount of adaptor molecules is captured at one end only and one end is available for sequencing.
Sequencing was performed as expected by the manufacturer of the instrument.
Example 2 (predictive) preparation of asymmetrically adjusted circular adaptor molecules for sequencing
In this experiment, a double-stranded target DNA was obtained. The DNA is fragmented to the appropriate size in vitro or naturally fragmented. Adaptors are hairpin molecules having a double stranded portion and a loop portion. Equal amounts of a mixture of the two adaptors are added. The adaptors differ at least in the loop sequence. Adapters are ligated to each end of the target DNA to produce adaptor molecules as described in Pacific Biosciences Template Preparation and Sequencing Guide (2012) Pacific Biosciences of California, Inc. and U.S. Pat. No. 8153375. See fig. 2A. The capture oligonucleotide is complementary to the single stranded portion of the adaptor molecule (FIG. 2B). The capture oligonucleotides also contained a poly-dA moiety complementary to a poly-dT oligonucleotide bound to polystyrene coated magnetic beads (DynaBeads, ThermoFisher, Waltham, Mass.) (see FIG. 2A). The capture oligonucleotide has several LNA bases that stabilize the complex with the adapter (see fig. 2A and 2B). Thus, another adaptor can be used for polymerase loading and sequencing initiation. The sequencing primer is complementary to the single-stranded portion of the adapter, and the sequencing polymerase is capable of extending the primer, thereby performing a sequencing reaction. The components were added in the order shown in figure 2. Sequencing was performed as expected by the manufacturer of the instrument.
Example 3 (predictive) preparation of asymmetrically adjusted Linear ligation molecules for sequencing
In this experiment, a double-stranded target DNA was obtained. DNA is fragmented to the appropriate size in vitro or each 3' -end of the naturally fragmented target DNA is extended with a terminal transferase and a single nucleotide (e.g., dATP) mixed with a small number of dideoxynucleotides (e.g., ddCTP). The target DNA has a homopolymer at each end. (FIGS. 4, 5) the capture oligonucleotide is directly bound to the solid support and is complementary to the homopolymer (e.g.with oligo-dT) (FIGS. 4, 5). The solid support included polystyrene coated magnetic beads (DynaBeads, ThermoFisher, Waltham, Mass.). The homopolymer can be used for polymerase loading and sequencing initiation.
To prevent two polymerases from loading on the same template (fig. 4), the 3' -end of the capture oligonucleotide was extended with dTTP and DNA polymerase, making it unusable for sequencing primers. The capture oligonucleotide was extended with a mixture of dTTP and ddTTP so that the extension product was not further extendable. Alternatively, the capture oligonucleotide is extended with dTTP and joined to the 5' -end of the target via ligation.
Example 4: workflow assembly of asymmetrically adjusted Linear junction molecules for sequencing
In this experiment, a series of assembly workflows was used to prepare sequencing libraries for sequencing according to the methods of the invention, as shown in fig. 8A-8F. The workflow was started with oligo-DT capture beads as described above (fig. 8A). The capture probe (SEQ ID NO:4) was added to the capture beads to form capture probe-capture bead complexes (FIG. 8B). A circular sequencing template comprising a double-stranded target nucleic acid molecule and two hairpin adaptors (hairpin adaptors A and B comprising SEQ ID NOS: 1 and 2, respectively) was contacted with the capture probe-capture bead complex (FIG. 8C) to form a complex of capture beads, capture probes, and circular sequencing template. Then, a sequencing primer (SEQ ID NO:5) (FIG. 8D) and a sequencing polymerase (FIG. 8E) were added. Figure 8F shows the results of analysis of the components of the assembled sequencing library when the components are added as shown in figures 8A-8E. All three were detected in the finally assembled complex only when the beads, capture probes and library molecules were all included.
Homopolymer only one of the two homopolymers is now available for polymerase loading and sequencing initiation. The sequencing primer is complementary to the homopolymer (e.g., has an oligo-dT), and the sequencing polymerase is able to extend the primer, thereby performing a sequencing reaction. The components were added in the order shown in figure 2. Sequencing was performed as expected by the manufacturer of the instrument.
Figure IDA0001948113260000011
Figure IDA0001948113260000021
Figure IDA0001948113260000031

Claims (17)

1. A method of determining the sequence of a double-stranded target nucleic acid in a sample, comprising:
(a) contacting a sample comprising a double-stranded target nucleic acid with a mixture of first and second hairpin adaptor molecules, each of the first and second hairpin adaptor molecules comprising a double-stranded stem region and a first or second single-stranded loop region;
(b) ligating each end of the target nucleic acid molecule to a double stranded region of an adaptor molecule, thereby forming a circular adaptor molecule comprising the target nucleic acid and having a double stranded region and a first single stranded loop region at one end and a second single stranded loop region at the other end covalently linked to the double stranded region;
(c) contacting the sample with a defined concentration of a blocking oligonucleotide tethered to a solid support, wherein the blocking oligonucleotide is not extendable by a nucleic acid polymerase and is complementary to the first single-stranded loop region, thereby capturing the circular molecule on the solid support and blocking the first single-stranded loop region;
(d) contacting the sample with an oligonucleotide primer complementary to a second single stranded loop region, thereby hybridizing the primer to the second single stranded loop region;
(e) extending the primer with a sequencing polymerase, thereby determining the sequence of the double stranded target nucleic acid.
2. The method of claim 1, wherein the ends of the target nucleic acid and the adaptor are ligated by enzymatic treatment.
3. The method of claim 2, wherein the enzymatic treatment is selected from the group consisting of nucleotide addition and digestion of the target nucleic acid and the adaptor with a restriction endonuclease.
4. The method of claim 1, wherein the blocking oligonucleotide is tethered to the solid support via a means selected from a covalent bond with a support molecule or a non-covalent bond with a support molecule.
5. The method of claim 4, wherein the non-covalent bond to the support molecule is a specific interaction selected from the group consisting of: hybridization of biotin-streptavidin, antibody-antigen or blocking oligonucleotides to complementary oligonucleotides covalently or non-covalently attached to a solid support.
6. The method of claim 1, wherein the blocking oligonucleotide is not extendable by a nucleic acid polymerase.
7. The method of claim 6, wherein the blocking oligonucleotide is rendered non-extendable by a nucleic acid polymerase by a chemical modification selected from the group consisting of 3' -H, 2' -phosphate, and 3' -phosphate.
8. The method of claim 1, wherein the blocking oligonucleotide has a modification that prevents binding of a nucleic acid polymerase.
9. The method of claim 1, wherein the blocking oligonucleotide is not extendable by a nucleic acid polymerase by virtue of being attached to the solid support via its 3' -end.
10. The method of claim 1, wherein the blocking oligonucleotide comprises one or more duplex stabilizing modifications.
11. The method of claim 10, wherein the duplex stabilizing modification is selected from the group consisting of Locked Nucleic Acid (LNA), Peptide Nucleic Acid (PNA), and non-natural nucleotides.
12. The method of claim 1, wherein the blocker oligonucleotide comprises one or more modifications that block nuclease digestion.
13. The method of claim 12, wherein the blocking oligonucleotide comprises a phosphorothioate backbone.
14. The method of claim 1, wherein the solid support is comprised of particles, and each particle is attached to a plurality of blocking oligonucleotides.
15. The method of claim 1, wherein extending the primer is by a strand displacing polymerase.
16. The method of claim 1, wherein the extension primer is replicated by rolling circle.
17. The method of claim 1, wherein the double stranded target nucleic acid is generated in vitro from a single stranded target nucleic acid.
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